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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2016  |  Volume : 59  |  Issue : 4  |  Page : 469-473
Assessment of antigen presenting cell infiltration in lung tissues of patients with bronchiectasis


1 Department of Surgery, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia
2 Department of Critical Care, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia
3 Department of Pathology and Immunology, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia

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Date of Web Publication10-Oct-2016
 

   Abstract 

Background: Bronchiectasis is a chronic disease characterized by permanent dilatation of the conducting airways accompanied by sustained inflammation. Aims: To assess whether chronic inflammation of lungs in bronchiectasis is associated with alterations in the numbers of infiltrating antigen presenting cell (APC). Setting and Design: Lobectomy specimens from 12 nonsmoker, nonasthmatic patients with acquired (noncongenital) bronchiectasis and six control patients were included in the study. Histopathology slides were reviewed, and immunohistochemical markers for dendritic cells (DCs) macrophages and Langerhans cells have been applied and analyzed. Materials and Methods: Tissue specimens were stained by immunohistochemistry using markers for DCs (CD83 and CD23), macrophages (CD68 and CD163), and Langerhans cells (CD1A and S-100 protein). The mean cell counts of stained cells in five high power microscopic fields were recorded. Statistical Analysis Used: Descriptive statistics, mean, standard deviation, median, and interquartile range were used. A nonparametric Mann-Whitney U-test was used to compare cell counts between bronchiectasis and control patients. P <0.05 was considered significant. Results: The mean age of patients with bronchiectasis and controls was 36.7 ± 16.6 and 31.8 ± 22.6 years, respectively. The predominant cell type among the patients was macrophage (median 50.5) followed by DCs (median 44.85), histiocytes (median 32), and Langerhans cells (median 5%). Compared to the controls a significantly higher number of macrophages (P = 0.01), DCs (P = 0.001), and Langerhans cells (P = 0.014) were present. Conclusion: Chronic inflammatory response in acquired (noncongenital) bronchiectasis is most probably mediated by increased infiltration of APCs in lung tissues.

Keywords: Dendritic cells, histiocytes, Langerhans cells

How to cite this article:
Hajjar WM, Alzeer AH, Fathaddin AA, Al-Otair HA, Al-Rikabi AC, Shakoor Z. Assessment of antigen presenting cell infiltration in lung tissues of patients with bronchiectasis. Indian J Pathol Microbiol 2016;59:469-73

How to cite this URL:
Hajjar WM, Alzeer AH, Fathaddin AA, Al-Otair HA, Al-Rikabi AC, Shakoor Z. Assessment of antigen presenting cell infiltration in lung tissues of patients with bronchiectasis. Indian J Pathol Microbiol [serial online] 2016 [cited 2019 Jun 16];59:469-73. Available from: http://www.ijpmonline.org/text.asp?2016/59/4/469/191779



   Introduction Top


Bronchiectasis is characterized by permanent dilatation of the bronchial airways due to repetitive chest infections. [1],[2],[3]

Although sustained inflammation in bronchiectasis in the absence of an obvious infectious cause. Furthermore, deregulated cytokine network which is independent of bacterial colonization has been reported previously, [4],[5],[6] the presence of activated T-cells expressing human leukocyte antigen-DR molecules in the lamina propria along with the presence of antigen presenting cells (APCs) such as dendritic cells (DCs) and macrophages suggest APCs driven immune responses mediating chronic inflammatory process associated with bronchiectasis. [7],[8],[9]

We hypothesize that repetitive airway infections with bacterial colonization in the lungs of patients with bronchiectasis is associated with increased infiltration of APCs. Using specific markers for each cell type, this study was performed to quantify infiltrating APCs in the lung tissues of patients with bronchiectasis.


   Materials and Methods Top


Patient population

Fifteen bronchial specimens were obtained from patients who underwent lobectomy for acquired (noncongenital) bronchiectasis between July 2009 and June 2014. The Institutional Review Board approved the study. All patients and controls previously gave written consent for the procedures. Specimens from three patients with a history of asthma, smoking, or using inhaled corticosteroids were excluded. Control specimens were obtained from six patients who had lobectomies for solitary fibrous tumor, carcinoid tumor, lung laceration, lung sequestration, spontaneous pneumothorax, and adenocarcinoma. The location of these tumors and lesions in control specimens was variable and included upper, middle, and lower lobes. The diagnosis of bronchiectasis was based on a high-resolution computed tomography (CT) scan of the chest (high-resolution CT) as previously described. [10] The sputum of all patients was cultured for ordinary bacteria and Mycobacterium tuberculosis (TB), and the sera of patients with bronchiectasis were tested for immunoglobulins and α-1 antitrypsin levels.

Histological analysis

Lung resection specimens obtained from patients with acquired (noncongenital) bronchiectasis, and control patients were cut into blocks, fixed in formalin, and embedded in paraffin. After dewaxing and microwave antigen retrieval, 5 μm-thick sections of lung parenchyma containing large airways were immunostained using commercially available immunochemistry kits (DAKO and Novocastra Ltd.) and 2 FDA-approved automated immunostainers (Vision Biosystem Ltd. Model: Bond Max and Ventena: Benchmark Xt). The panel of monoclonal antibodies included CD83, CD23, CD68, CD163, CD1A, and S-100 protein [11] (DAKO and Ventana Inc. Ltd.).

Sections obtained from the lung resection specimens were coded and positively stained cells were counted in a blinded fashion by two observers (AAF and ACAR) using a Nikon Eclipse 80i microscope with an eyepiece graticule containing 100 equidistant points at a magnification of 200. Immunopositive cells in large airways having supporting cartilage with a nucleus visible in the section plane were counted in the entire area of the section beneath the basement membrane. We used a validated point-counting technique as described by other investigators. [12],[13] All areas covering five high power fields of the bronchial epithelium and subepithelium, including areas within the smooth muscle layers and perivascular spaces, were counted and analyzed by both observers. Nucleated cells showing dendritic or histiocytic morphology with membrane and/or cytoplasmic staining were regarded as positive; however, for S-100 protein, only cells showing both nuclear and cytoplasmic staining were counted as positive. The coefficients of variation (CV) were <5% for repeated cell counts by the same observer for each antibody and interobserver CVs were <10% for all antibodies. Good correlation (P ≤ 0.001) was achieved between the two observers (ACR and AAF). Results from sections obtained from each representative block (one paraffin block per patient and control) were averaged and tabulated.

Statistical analysis

Descriptive statistics, mean, standard deviation, median, and interquartile range were used. A nonparametric Mann-Whitney U-test was used to compare cell counts between bronchiectasis and control patients. P < 0.05 was considered significant.


   Results Top


Of 12 patients with bronchiectasis, seven (58.3%) were females and two (33.3%) out of six control subjects were females. Sputum cultures from five (41.7%) bronchiectasis patients had normal respiratory flora while three were positive for Haemophilus influenzae two for Streptococcus pneumoniae, and two for Moraxella catarrhalis. The sputum of control patients had normal respiratory flora, but one grew S. pneumoniae. All sputum specimens in both groups were negative for TB and patients with bronchiectasis had normal levels of immunoglobulins and α-1 anti-trypsin.

[Table 1] describes data for quantification and comparison of different cell types detected in the lung tissues of patients with bronchiectasis and controls. The mean ranks of CD83 positive cells in patients with bronchiectasis (12.50) was significantly higher (P - 0.01) than the controls (3.50) as P < 0.05 was considered significant. Whereas CD23 positive cell was detected in control lung biopsies with mean rank of 3.50 and the mean rank of CD23 cells in patients with bronchiectasis was 12.50 (P - 0.001). The mean rank of CD68 positive cells in patients with bronchiectasis (11.75) was significantly greater than the median number of CD68 cells (5.0) present in the controls specimens (P - 0.01). There was no statistical difference in the mean ranks of CD163 in the biopsy samples from patients with bronchiectasis (9.50) and controls (9.50). The mean ranks of cells expressing CD1A were more in the lung tissues from bronchiectasis patients (11.67) compared with controls (5.17) and (P - 0.014). The mean ranks of S-100 protein-positive cells in the lung biopsies from patients with bronchiectasis (10.5) were not statistically different than the controls (7.50).
Table 1: Comparison of antigen presenting cells in lung tissues of patients with bronchiectasis and controls

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Histological examination of the bronchiectatic tissue showed relative preservation of the bronchial and bronchiolar epithelium with only occasional areas of ulceration. Mononuclear cells were seen infiltrating the cells of the pseudostratified epithelium. The most striking and constant feature was the inflammatory cell infiltration of the basement membranes, submucosal tissue, and sometimes muscularis [Figure 1]a. There was also evidence of increased vascularity, mainly in the submucosa; however, the bronchial cartilage was well preserved. There were relatively few polymorphonuclear neutrophils in the bronchial and bronchiolar walls. Neutrophils were, however, identified in the lumen of some bronchi and bronchioles in association with mucus and cellular debris and mature lymphocytes.[Figure 1]b DCs identified as brown stained cells expressing CD83 marker were located mainly in subepithelial region of the airway wall in both lamina propria and adventitia [Figure 1]c. CD23 positive cells were located in the perifollicular tissues [Figure 1]d. A homogeneous pancytoplasmic positive staining was observed for CD68 marker representing macrophages [Figure 2]a. Cells containing kidney-shaped nuclear outline with histiocytic morphology expressing CD163 marker were observed in the submucosal layers [Figure 2]b. Similarly, cells with morphological appearance of DCs labeled with CD1A marker were also present in the submucosal tissues [Figure 2]c. S-100 protein positive cells were located in the intraepithelial layer, with dark brown staining of both the cytoplasm and nucleus [Figure 2]d.
Figure 1: (a) Increased number of intraepithelial neutrophils and subepithelial mononuclear cells (arrowhead) in bronchiectasis bronchi. (b) Mononuclear inflammatory cell infiltration including many mature lymphocytes (arrowhead). (c) CD83 positive cells located within the basal and subepithelial layers of bronchial tissues. (d) CD23 positive cells located in the perifollicular tissues

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Figure 2: (a) Numerous CD68 positive histiocytes are present in the subepithelial connective tissue of the major bronchus. (b) CD163 positive histiocytes in the submucosal layer of a major bronchus with kidney - shaped nuclear outline. (c) Positive CD1A stained cells (arrowhead) in the bronchial submucosal tissues. (d) Dark brown stained S - 100 protein positive cells (arrowhead) located in intra - epithelial layer

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   Discussion Top


Our results demonstrate increased infiltration of cells expressing markers for DCs and macrophages in the large airways of patients with bronchiectasis. The findings of the present study are consistent with a previous study performed by Silva et al., who documented an increase of DCs infiltration in the epithelium, lamina propria, and submucosa of the airways in patients with bronchiectasis. The number of patients in our study is, however, larger. In addition Silva et al. emphasized more on the role of various T-lymphocytes in the pathogenesis of bronchiectasis than DCs and other APC's as it is the case in this study. [9] A predominant mononuclear cell infiltration including DCs in the lungs particularly in the bronchial walls and around blood vessels was also reported in rat model of bronchiectasis. [14] Pulmonary DCs reside in the lung parenchyma and airways and provide effective surveillance against inhaled allergens, viruses, and bacteria invading the mucosal surface of the bronchi and bronchioles performing a key role in innate immune responses. [15],[16] In addition, DCs serving as APCs also serve as a vital link between the innate and adaptive immune response. [15],[17] The presence of higher numbers of DCs in patients with bronchiectasis detected in this study along with increased numbers of activated lymphocytes may, therefore, be a reflection of simultaneous participation of DCs in both innate and adaptive immune responses in bronchiectasis mediating a sustained inflammatory process resulting in airway remodeling.

The exact pathogenesis of the airway destruction in these patients remains to be fully elucidated. Cole proposed a vicious cycle of microbial infection, airway inflammation, and tissue damage, incriminating both local and systemic inflammation in the disease process. [18] Furthermore, increasing evidence supports that bacterial and/or viral infection modulates immunity by altering several immune competent cells, including DCs. [16] In this process, DCs are localized to initiate an inflammatory reaction in response to bacterial infection. In fact, antigen presentation has been shown in models of bronchiectasis. A rapid influx of DCs expressing cell surface major histocompatibility complex antigens in a rat model following airway exposure to M. catarrhalis earlier then neutrophil influx highlights the pivotal role of pulmonary DCs in defending against the invading microbes. [19] The lower lobe airways of patients with bronchiectasis are known to harbor bacterial pathogens due to alteration in airway homeostasis and the presence of mature DCs in virtually all the mucosal tissues examined in this study could possibly be DCs response to microbial presence in the bronchiectatic lungs. [20] Although over half of the patients in this study had pathogens in their sputum, it is however difficult to link the bacterial presence with increased infiltration of DCs.

The number of macrophages infiltrating the lung tissues of patients with bronchiectasis was significantly higher than controls in the present study. This observation is in agreement with the previously reported finding of higher numbers of CD68+ macrophages detected in lamina propria of patients with bronchiectasis. [21],[22] As opposed to neutrophils little is known about the exact role of macrophages in bronchiectasis. Neutrophils are frequently detected in lungs of patients with bronchiectasis and macrophages contribute significantly in neutrophil influx by releasing tumor necrosis factor-α. [22] Macrophages also influence neutrophil infiltration by releasing IL-8. [23] Because of the higher influx of neutrophils in their lung tissues, patients with bronchiectasis also tend to harbor a higher number of apoptotic neutrophils resulting in tissue damage and persistent inflammation. [24] Ineffective removal of apoptotic cells by macrophages may further complicate the resolution phase and may have a detrimental effect on the airways. [25] Macrophages release tissue metalloproteinase, an enzyme capable of degrading collagen IV and may contribute in the remodeling of lung tissue. [26] Furthermore, release of metalloproteinases may also help in restoration of the impaired migration through tracheal epithelial tight junctions by metalloproteinase-9 deficient DCs. [27] Therefore, the presence of high numbers of macrophages in the lungs of our patients with bronchiectasis suggests that macrophages may be serving as regulatory cells in the ongoing inflammatory processes associated with brochiecatasis.

No difference in the number of cells expressing CD163 was found among the patients with bronchiectasis and the controls. Although a marker for histiocyte differentiation CD163 is also expressed on monocytes/macrophages. [28] CD1A positive cells in lung tissue specimens from patients with bronchiectasis were present in a significantly greater numbers compared to the controls in the present study. These cells are usually present in the epithelial layer of the airway. [29] CD1A, a marker for Langerhans cells, is also expressed on immature DCs eventually maturing into CD83+ DCs. [30] S-100 proteins marker is considered as a specific marker for Langerhans cells and their derivatives, but it can be detected on a variety of cells such as lymphoid tissue, adipocytes, cartilage, and some myoepithelial cells. [31] Collectively varying numbers of cells expressing the different markers in this study represent various differentiation phases of cells belonging to DCs and macrophage lineages supporting preponderance of APCs activity in patients with brocnchiectasis.


   Conclusion Top


Higher numbers of APCs infiltrating the lung tissues in nonsmoker, nonasthmatic, patients with acquired (noncongenital) bronchiectasis indicate heightened adaptive immune responses. Chronic inflammation associated with bronchiectasis may possibly be due to persistent immune activation as result of the presentation of yet unknown antigen/s leading to remodeling of pulmonary tissues. Although pathogens were isolated from the sputum of majority of the patients with bronchiectasis, the study, however, fell short of establishing a direct link of respiratory tract infections with increased numbers of APCs detected in the lung tissues. In addition, studies are indicated to validate the findings of the present study and further characterization of the DCs subsets in bronchiectasis.

Furthermore, at present, there are no clinical applications for the findings of this study but anti-dendritic or APC's agents could be researched and probably used in the future.

Acknowledgments

This study was supported by the College of Medicine Research Center, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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Correspondence Address:
Waseem M Hajjar
Department of Surgery, College of Medicine, King Saud University, P.O.Box 7805, Riyadh 11472
Kingdom of Saudi Arabia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.191779

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